The proposed research will include the following: (1) Ongoing studies of chemical modification of allosteric sites of rabbit skeletal muscle phosphofructokinase (PKF) will be extended. These studies will examine the kinetic regulatory properties of PFK modified at the AMP binding site and of PFK hybrids containing native enzyme and enzyme with either a modified reactive thiol group, a modified citrate binding site, a modified ATP inhibitory site, or a modified AMP site. PKF modified with fluoresecent derivatives will be studied to determine protein conformational changes due to ligand binding and to study intra- and interprotomeric distances between different probes on the molecules by means of fluorescence energy transfer measurements; (2) A study of the characterization, distribution, and physiological role of multiple isozymic forms of PFK will be continued employing electrophoretic and immunochemical techniques. Emphasis will be placed on the yet to be characterized C isozyme and its regulatory properties. In addition, we will examine possible adaptive responses by brain, liver, and adipose tissue PFKs to varying dietary and hormonal states; and (3) The regulatory significance of phospho- and dephospho-forms of muscle PFK will be investigated.